Novel calcium-sensitive ligand-receptor interactions have been sought for further investigation of calcium-mediated signal regulation. The A2058 human melanoma cell line has been chosen. Calcium-influx responses have been documented when these cells are bathed with carbachol, the muscarinic acetylcholine agonist, ATP, agonist of the P2Y receptors, and thrombin. The observations of the carbachol-induced activity have been characterized and have demonstrated that there is a low abundance, high affinity muscarinic receptor on in the membrane of A2058 cells. When activated, this receptor stimulates calcium influx and intracellular release, release of arachidonic acid, and inhibition of cAMP production. No generation of inositol phosphates has been demonstrated using three different assay systems. Biologic behavior for this receptor has been demonstrated by its inhibitory action on the soft agar colonization of A2058 cells in the presence of carbachol which is completely abrogated by coincubated with atropine. Molecular characterization of this receptor suggests that it may have similarity to the m2 subtype by Northern analysis and the m3 subtype by pharmacologic analysis. PCR and detailed pharmacologic mapping are ongoing.